The overall objective of this proposal is to determine the significance of epigenetic histone post-translational modifications (PTMs) during human cytomegalovirus (HCMV) infection. HCMV is the largest known human herpes virus and it is well recognized that a large percentage of the human population (>60%) is infected with HCMV. HCMV infections cause birth defects; and for immunocompromised populations, these infections are often times life threatening. Upon infection of cells and chromatinization of the virus in the host nucleus, the major immediate-early promoter (MIEP) controls the expression of the viral IE1 and IE2 proteins which interact with many nuclear proteins. These interactions ultimately modulate both viral and cellular gene expression, in part through epigenetic mechanisms. Epigenetics refers to mechanisms that can regulate gene expression patterns without involving changes in DNA sequence, and include DNA methylation, small ncRNAs and histone PTMs. The limited picture of how HCMV harnesses epigenetic mechanisms presents a significant barrier to understanding HCMV pathogenesis and developing novel antiviral therapy to target these chromatin factors. Previously, we determined that HCMV infection results in several changes to single PTM sites on histones, and that one modification site in particular H3K79me2 and its corresponding enzyme DOT1L affected HCMV growth. Here we aim to answer the following questions: How does HCMV utilize combinatorial PTMs on histones for controlling both host and viral gene expression patterns and viral replication? Are there any non- histone chromatin proteins that are needed for viral growth? As certain protein lysine methyltransferases are upregulated during HCMV infection, are there other non-histone proteins methylated during infection that are needed for viral replication? We will address these questions using a combination of biological and quantitative methods. We will develop enhanced mass spectrometry methods to quantify combinatorial histone H3 and H4 PTMs from MRC5 cells during an HCMV infection time-course. Genome-wide approaches will be employed to identify both host and viral genes harboring significant combinatorial PTMs. The effect of depleting the enzymes responsible for the most abundant combinatorial PTM changes on both host and viral gene expression, and on viral growth and replication will be assessed. Using biochemical and affinity approaches, we will isolate both global viral chromatin and MIEP specific viral chromatin to determine the chromatin factors that contribute to regulation of viral gene transcription. Lastl, we will use our novel affinity quantitative proteomics approach to identify non-histone proteins that are lysine methylated during viral infection. It is expected that these experiments will produce new fundamental insights in an understudied area of HCMV biology, and could lead to development of new epigenetic drug therapy for treatment of HCMV.